1. Field of the Invention
This invention relates to a process and device for determining the electrophoretic mobility of fluorophore labeled nucleotide particles in gels by detecting fluorophore movement after photobleaching utilizing interference pattern produced by crossed laser beams or differaction gratings having spacing in micron dimensions.
2. Background of the Related Art
Conventional gel electrophoresis has been a powerful analytic method for DNA separations. However, it is limited for effective fractionation of large DNA fragments up to about 20 kilo base pairs (kbp). Recently, Schwartz and Cantor, in Cell, 37, 67-75 (1984) introduced a pulsed-field gel electrophoresis (PFGE) technique, which allows separation of DNA molecules up to 2 mega base pair (Mbp). Modifications to the geometry of the applied electric field and other considerations to this technique have further improved the size resolution limit to 5.about.10 Mbp. See, Carle, et al., Science, 232, 65-68 (1986). The progress in PFGE electrophoresis has been reviewed by Cantor, et al. in Ann. Rev. Biophys. Biophys. Chem., 17, 287-304 (1988).
In PFGE electrophoresis, one critical experimental parameter is pulse width (i.e., the time an electric field is applied in one direction before it is switched off or changed to another direction) which sets an upper limit on the DNA separation range. As pulse width approaches the DNA reorientation time, the electrophoretic mobility changes sharply with the molecular weight, and thus high resolution for DNA separation is achieved. Besides the pulse width, the separation resolution also depends on many other parameters, such as field strength, gel structure, DNA conformation and effective charge; other parameters include the geometry of the applied electric field, and the temperature. Therefore, if the parameters could be tuned to an optimum condition, the resolution could be improved and an even higher separation limit could be achieved. As a smallest human chromosome is estimated to be 30 Mbp (see, Anand, Trends Genet., 2 278-283 (1986)) it appears that PFGE electrophoresis could become one of the major analytical tools for the human genome project. However, the details of the fundamental mechanism in this very important new technique remain semipractical at best. The parameters that influence the PFGE separation are numerous and coupled in a complex manner. Optimization of PFGE requires a thorough understanding of the dynamics of DNA chain deformation and corresponding electrophoretic motion in gels under an applied electric field. Chu et al. in Biopolymers, 27, 2005-2009 (1988), have studied orientation and stretching times of large DNA fragments in agarose gels by low-field electric birefringence (TEB). The low-field, long pulse width electric field birefringence measurements, however, could only provide information on DNA conformation dynamics including chain orientation and stretching.
A biased reptation model, as described by Lerman et al., Biopolymers. 21, 995 (1982); Lumpkin et al., Biopolymers, 21, 2315 (1982); Lumpkin et al., Biopolymers, 24, 1573 (1985); Slater et al., Phys. Rev. Lett., 55, 1579 (1985), Biopolymers, 25, 431 (1986); Noolandi, et al., Phys Rev. Lett., 58, 2428 (1987); and Hervet et al., Biopolymers, 26, 727 (1987), has been applied successfully to conventional gel electrophoresis (GE). Reptation theories have been further extended to include chain fluctuations; see Zimm, Phys. Rev. Lett., 61, 2965 (1988); Noolandi et al., Science, 243, 1456 (1989). Recently, a numerical simulation proposed by Deutsch, in Science, 240, 922 (1988); J. Chem. Phys., 90, 7436 (1989); and Deutsch et al., J. Chem. Phys., 90, 2476 (1989), has provided a more detailed insight about DNA motion during PFGE and conventional GE. The suggested elongation-contraction chain behavior in an applied electric field has been used to explain the overshoot-undershoot phenomenon in fluorescence detected linear dichroism measurements; see Holtzwarth et al., Nucleic Acids Res., 15, 10031 (1987). Observation of conformational changes of individual DNA molecules during gel electrophoresis by fluorescence microscopy supported the simulation results found by Deutsch; see, Smith et al. Science. 243, 203 (1989); and Schwartz et al., Nature (London), 338, 520 (1989). Few directed experimental measurements have been reported, however, on the time-dependent electrophoretic mobility of DNA in gels while the DNA chains are being deformed, for example see, Holtzwarth et al., Biopolymers, 28, 1043 (1989).
Fluorescence recovery after photobleaching (FRAP) has been used for at least 15 years. Modifications to this method, including photobleaching a pattern instead of a simple spot on the sample and modulation detection of the fluorescence recovery signal, have been introduced to improve the ease with which FRAP can be applied to a variety of problems. See, for example, Smith & McConnell Proc. Natl. Acad. Sci. USA, 75, 2759-2766 (1978); Lanni & Ware, Rev. Sci. Instrum., 53, 905-908 (1982); and, Wahl, Biophys. Chem., 22, 317-321 (1985). Presently, FRAP has already become a common technique for measuring the mobility of specific components in complex systems, especially for the measurements of lateral mobility of lipid bilayers and of proteins in cell plasma membranes and cell organelle envelopes, as reported by Peters et al., Biochim. Biophys. Acta., 367, 282-287 (1974); Axelrod et al., Biophys. J., 16, 1055-1069 (1976); Jacobson et al., Biochim. Biophys. Acta, 433, 215-222 (1976); and, Koppel et al., Biophys. J., 16, 1315-1325 (1976).
Diffusion and interaction of macromolecules in solution, as well as molecular motions in the cytoplasm and nucleoplasm can also be studied using FRAP. The idea for FRAP is simple and clear. The molecular species of interest are either fluorophores or molecules labeled with a fluorophore. Mobility of the fluorophores or of the fluorophore-labeled molecules is then measured by bleaching a spot (or pattern) on the sample with an intense pulse of light. The time for the fluorescence recovery, i.e., the dissipation of the bleached pattern, is a function of the size of the bleached area and the rate of mobility of the fluorophores or labeled molecules. The periodic pattern of photobleaching technique invented by Smith and McConnell (1978) supra., makes FRAP simpler in theory and in practice. The periodic pattern can be obtained either by using a diffraction grating (such as a Ronchi ruling) or by using two coherent crossed laser beams to produce an interference pattern. Two important aspects of the periodic pattern technique are its insensitivity to deviation of the bleached pattern from a pure spatial sinusoid, and its usefulness in detection of anisotropic diffusion in the image plane; see, Lanni et al., Biophys. J., 35, 351-358 (1981).
U.S. Pat. No. 4,222,744 to McConnell describes in general terms an assay for label ligands such as fluorescent labeled nucleotides in which the mobilities of the ligands are detected by observing fluorescence recovery after photobleaching of the labeled ligands. The ligands are bleached using a single laser focused to a spot in a fluid or gel or using a laser generated pattern of lines.
U.S. Pat. No. 4,675,095 to Kambara describes an electrofluoretic apparatus for detecting electrofluoretic labeled nucleotides after excitation by a single light source, such as a laser. The disclosure is specifically directed to the production of low background noise by exciting the fluorophore labeled nucleotides by incident excitation light projected in the gap between the two glass plates substantially parallel to the boundary planes of the gel. Fluorescence is detected in a direction perpendicular to the excitation light source.
Similarly, U.S. Pat. No. 4,832,815 to Kambara et al. cites the Smith et al. article in Nature (1986) supra., for the disclosure of a system in which a DNA fragment is detected in real time during electrophoresis separation through fluorescent labeling of the DNA. The DNA fragments are irradiated with a laser and when they pass through the irradiated region they give forth fluorescence successively from the shortest fragment. Since the emission wavelengths differ depending upon the base species, the base species are determined from the wavelengths. The lengths of the frame can thus be determined from the migration times.
Likewise, U.S. Pat. No. 4,832,815 describes a wavelength dispersion electrophoresis apparatus which detects fluorescence of unequal wavelengths emitted from samples of nucleotides which are labeled with a plurality of fluorophores. A single laser beam is used to excite the fluorophores at a particular location in the gel while a direct-vision prism is interposed between the two dimensional fluorescence detector and electrophoretic plate in order to separate and discriminate the emission wavelengths of the respective fluorophores.
Capillary electrophoresis has been used to detect the separation of nucleic acids in a very small volume, as reported by Kasper et al., J. Chromatogr., 458, 303-312 (1988). Kasper et al. describe experiments using absorbance and fluroscence detectors modified for use with a 50-100 .mu.m inside diameter (I.D.) capillary tubing. The instruments were tested, and a signal to noise ratio of 3 was observed in measuring 15 .mu.g/ml for fluorescense detected of ethidium bromide-stained herring-sperm DNA and 3 .mu.g/ml for absorbance detection, at a separation voltage of up to 30 kV.
Electrophoretic mobilities of photochromically labeled ions have been measured by combining electrophoresis with holographic relaxations spectroscopy (HRS) or forced Rayleigh scattering (FRS), as reported by Rhee et al., J. Phys. Chem., 88, 3944-3946 (1984); and Kim et al., J. Phys. Chem., 88, 3946-3949 (1984). The species of interest in HRS or FRS, however, have to be photochromic or labeled with photochromic dyes whose lifetimes are longer than the relaxation time of interest. Unfortunately, in differentiating and determining the dynamics of large DNA fragments, the lifetimes of most photochromic dyes are too short when compared with the time required to measure the slow DNA electrophoretic mobility in gels. The related art, therefore, does not describe techniques for very rapidly measuring the electrophoretic mobility and dynamics of large DNA particles.
Accordingly, it is an object of the present invention to provide a technique for very rapidly determining the mobility of polyelectrolytes, especially large DNA fragments in gels.